A cell therapy capable of sustained therapeutic protein delivery in vivo has the potential to benefit both kidney disease and its complications. During our previous grant cycle, we developed and validated technology using transposon-modified antigen specific T cells for therapeutic protein delivery in vivo using erythropoietin (EPO) as a model system. We demonstrated delivery of murine EPO and therapy for anemia of chronic kidney disease in mice as a model system, and we demonstrated inducible human EPO expression from antigen-specific human T cells in vitro. We propose to significantly advance beyond our previous grant by extending our studies to human T cells in an in vivo context and testing an innovative approach to enhance long-term therapeutic enzyme delivery for Fabry disease that results from loss of ?-galactosidase A (?-gal A). ?-gal A -/- mice represent an animal model of human Fabry disease, which results from lack of an enzyme needed to metabolize fats leading systemic disease including kidney disease. In aim 1, we will test transposon-modified antigen-specific T cells for expression of ?-gal A in a Fabry disease model. We will extend our mouse studies with EPO to ?-gal A in a Fabry disease model using antigen-specific mouse T cells and vaccination. We will gene modify human T cells to express a chimeric antigen receptor (CAR) along with luciferase or human ?-gal A. Cells will be infused into NOD/SCID/Fabry mice to evaluate the ability of engineered antigen-specific human T cells to engraft, respond to vaccination, and short-term expression ?-gal A in an in vivo model. Although perforin is important in T cells for clearance of malignant cells, the perforin pathway also contributes to clearance of antigen expressing cells post vaccination. In aim 2, we will test perforin knockout in T cells as a way of enhancing long-term therapeutic protein delivery from antigen-specific T cells. We propose to test perforin knockout in the setting of antigen- specific T cells to determine if it will boost long-term persistence of cells delivering therapeutic proteins. In aim 3, we will test transposon-modified antigen-specific T cells for long-term expression of ?-gal A in a mouse model of Fabry disease. We propose to deliver optimized human CAR-T cells expressing human ?-gal A in a NOD/SCID/Fabry mouse model. We will evaluate for engraftment, vaccination response, ?-gal A activity, and globotriaosylceramide levels in tissues. The proposed studies will lead to the development of new cell therapies for kidney disease and its complications and have the potential for therapeutic impact well beyond the kidney.